A diverse range of glycoconjugates exists in nature. See, e.g., Varki et al., Essentials of Glycobiology, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2009. These glycoconjugates play fundamental roles in cell structure, signaling processes, and cell-cell recognition, but studying them is challenging due to a lack of suitable chemical tools. See, e.g., Kiessling et al., Annu. Rev. Biochem. (2010) 79:619-653. Glycosyltransferases (Gtfs) are the enzymes that assemble these glycoconjugates from carbohydrate building blocks. See, e.g., Wagner et al., Chembiochem. (2010) 11:1939-1949; Brown et al., Crit. Rev. Biochem. Mol. Biol. (2007) 42:481-515; Pesnot et al., Nat. Chem. Biol. (2010) 6:321-323; and Frantom et al., J. Am. Chem. Soc. (2010) 132:6626-6627. Most Gtfs transfer a sugar from an anionic leaving group, for example, a nucleotide, to an acceptor such as another sugar, a protein, or a lipid. See, e.g., Lairson et al., Annu. Rev. Biochem. (2008) 77:521-555.
O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) catalyzes the addition of a single N-acetylglucosamine in O-glycosidic linkage to serine or threonine residues of intracellular proteins. OGT is an essential vertebrate Gtf that β-O-GlcNAcylates a wide variety of nuclear and cytoplasmic proteins, including transcription factors, cytoskeletal proteins, metabolic enzymes, kinases, phosphatases, proteasome components, chaperones, and neural proteins. See, e.g., Gambetta et al., Science (2009) 325:93-96; Sinclair et al., Proc. Natl. Acad. Sci. USA (2009) 106:13427-32; Love et al., Proc. Natl. Acad. Sci. USA (2010) 107:7413-18. OGT-mediated glycosylation is dynamic. A corresponding glycosidase, O-GlcNAc hydrolase (OGA), removes O-GlcNAc residues from proteins. See, e.g., Goldberg et al., Endocrinology (2006) 147:222-31; Kreppel et al., J. Biol. Chem. (1997) 272:9308-15. O-GlcNAc cycling is sensitive to stress conditions and nutrient status, particularly glucose levels. See, e.g., Lubas et al., J. Biol. Chem. (1997) 272:9316-24. OGT glycosylates many aminoacid side chains that could otherwise be phosphorylated, suggesting that OGlcNAcylation modulates kinase signaling. See, e.g., Jinek et al., Nat. Struct. Mol. Biol. (2004) 11:1001-07; Ha et al., Protein Sci. (2000) 9:1045-52; Hu et al., Proc. Natl. Acad. Sci. USA (2003) 100:845-849. Excessive OGT activity results in hyper-OGlcNAcylation, which is correlated with widespread transcriptional changes and a number of pathologies, including cancer. See, e.g., Wrabl et al., J. Mol. Biol. (2001) 314:365-74; Martinez-Fleites et al., Nat. Struct. Mol. Biol. (2008) 15:764-65.
Small molecule inhibitors of OGT have been sought for many years as cellular probes, and different approaches to identify such inhibitors have been explored. See, e.g., Gloster et al., Nat. Chem. Biol. (2011) 7:174-181; Konrad et al., Biochem. Biophys. Res. Commun. (2002) 293:207-212; Hajduch et al., Carbohydr. Res. (2008) 343:189-195. OGT has been found to be a challenging target because it is a nucleotide-sugar glycosyltransferase and the donor sugar substrate contains a diphosphate leaving group. The proposed transition state for these types of enzymes is dissociative, and the oxonium ion-like portion resembles the transition state of glycosidases. See, e.g., Vocadlo et al., Curr. Opin. Chem. Biol. (2008) 12:539-555; Gloster et al., Org. Biomol. Chem. (2010) 8:305-320. A key difference in glycosidase and Gtf transition states is that in the latter a negatively charged diphosphate leaving group rather than a carboxylate side chain helps stabilize the oxonium character. See, e.g., Lairson et al., Annu. Rev. Biochem. (2008) 77:521-555. Accordingly, although there are many good inhibitors of glycosidases, including OGA, these compounds typically do not inhibit Gtfs effectively. See, e.g., Macauley et al., Biochim. Biophys. Acta (2010) 1800:107-121; Rempel et al., Glycobiology (2008) 18:570-586; Dorfmueller et al., Chem. Biol. (2010) 17:1250-1255; Kim et al., J. Am. Chem. Soc. (2006) 128:4234-4235.
Efforts to identify selective Gtf inhibitors have focused primarily on the design of substrate mimics of negatively charged diphosphates. See, e.g., Trunkfield et al., Bioorg. Med. Chem. (2010) 18:2651-2663; Izumi et al., Curr. Top. Med. Chem. 9, 87-105 (2009); Skropeta et al., Glycoconj. J. (2004) 21:205-219. A major hurdle has been finding suitable replacements for the anionic phosphates. See, e.g., Wang et al., Bioorg. Med. Chem. (1997) 5:661-672; Helm et al., J. Am. Chem. Soc. (2003) 125:11168-11169; Hang et al., Chem. Biol. (2004) 11:337-345. These phosphates contribute significantly to binding affinity, and replacing them with neutral linkers usually results in weak inhibitors. Furthermore, retaining the phosphates typically prevents the Gtf inhibitor from getting into a cell. Vocadlo and coworkers developed protected sugar analogs of Gtf inhibitors which were fed to cells and subsequently metabolized into the corresponding nonhydrolyzable nucleotide-sugar donors in vitro. See, e.g., Gloster et al., Nat. Chem. Biol. (2011) 7:174-181. The method of Vocadlo allows polar donor analogs to be used as inhibitors in cells but it offers limited opportunities to tune selectivity since the inhibitors produced closely resemble common cellular substrates. Thus, there continues to remain a need for new and alternative approaches in the development of Gtf inhibitors, particularly OGT inhibitors, for use in studying the role of glycosylation in the cell as well as in the treatment of diseases associated with aberrant glycosylation.
Another important class of enzymes that could be inhibited with diphosphate mimetics is protein kinases. Protein kinases constitute a large family of structurally related enzymes that are responsible for the control of a variety of signal transduction processes within the cell. Protein kinases are thought to have evolved from a common ancestral gene due to the conservation of their structure and catalytic function. Almost all kinases contain a similar 250-300 amino acid catalytic domain. Kinases may be categorized into families by the substrates they phosphorylate (e.g., protein-tyrosine, protein-serine/threonine, lipids, etc.). In general, protein kinases mediate intracellular signaling by effecting a phosphoryl transfer from a nucleoside triphosphate to a protein acceptor that is involved in a signaling pathway. These phosphorylation events act as molecular on/off switches that can modulate or regulate the target protein's biological function. These phosphorylation events are ultimately triggered in response to a variety of extracellular and other stimuli. Examples of such stimuli include environmental and chemical stress signals (e.g., osmotic shock, heat shock, ultraviolet radiation, bacterial endotoxin, and H2O2), cytokines (e.g., interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α)), and growth factors (e.g., granulocyte macrophage-colony-stimulating factor (GM-CSF), and fibroblast growth factor (FGF)). An extracellular stimulus may affect one or more cellular responses related to cell growth, migration, differentiation, secretion of hormones, activation of transcription factors, muscle contraction, glucose metabolism, control of protein synthesis, and regulation of the cell cycle.
Many diseases are associated with abnormal cellular responses triggered by protein kinase-mediated events as described above. These diseases include, but are not limited to, autoimmune diseases, inflammatory diseases, neurodegenerative diseases, and proliferative diseases (e.g., cancer). Accordingly, there remains a need to find protein kinase inhibitors useful as therapeutic agents.